Cancer patients with clonal hematopoiesis die from primary malignancy or comorbidities despite higher rates of transformation to myeloid neoplasms

Abstract Background The occurrence of somatic mutations in patients with no evidence of hematological disorders is called clonal hematopoiesis (CH). CH, whose subtypes include CH of indeterminate potential and clonal cytopenia of undetermined significance, has been associated with both hematologic cancers and systemic comorbidities. However, CH's effect on patients, especially those with concomitant malignancies, is not fully understood. Methods We performed a retrospective evaluation of all patients with CH at a tertiary cancer center. Patient characteristics, mutational data, and outcomes were collected and analyzed. Results Of 78 individuals included, 59 (76%) had a history of cancer and 60 (77%) had moderate to severe comorbidity burdens. DNMT3A, TET2, TP53, and ASXL1 were the most common mutations. For the entire cohort, the 2‐year overall survival rate was 79% (95% CI: 70, 90), while the median survival was not reached. Of 20 observed deaths, most were related to primary malignancies (n = 7, 35%), comorbidities (n = 4, 20%), or myeloid neoplasms (n = 4, 20%). Twelve patients (15%) experienced transformation to a myeloid neoplasm. According to the clonal hematopoiesis risk score, the 3‐year transformation rate was 0% in low‐risk, 15% in intermediate‐risk (p = 0.098), and 28% in high‐risk (p = 0.05) patients. By multivariate analysis, transformation was associated with variant allele frequency ≥0.2 and hemoglobin <10 g/dL. Conclusions In a population including mostly cancer patients, CH was associated with comorbidities and myeloid transformation in patients with higher mutational burdens and anemia. Nevertheless, such patients were less likely to die of their myeloid neoplasm than of primary malignancy or comorbidities.


| INTRODUCTION
The presence of somatic mutations in a subpopulation of hematopoietic cells in individuals without a morphological diagnosis is known as clonal hematopoiesis (CH).3][4][5] In addition, the presence of pre-leukemic CH prior to cancer therapy is common in patients, who later develop therapy-related myeloid disorders, [6][7][8][9] a phenomenon likely related to increased fitness of the underlying CH. 10 However, no guidelines exist for hematologic monitoring, comorbidity mitigation, and leukemia prevention in patients with CH.Furthermore, the impact of CH on the outcomes of patients with concomitant underlying malignancies remains unknown.To gain insight into the natural history of CH in this patient population, we analyzed the characteristics and outcomes of a cohort of patients with CH from a single tertiary cancer center.

| Study design and participants
In this retrospective analysis, we evaluated the characteristics and outcomes of a cohort of patients with CHIP and CCUS actively being observed at The University of Texas MD Anderson Cancer Center.Included were patients in whom one or more somatic mutations was found by nextgeneration sequencing (NGS) of their bone marrow or peripheral blood from January 2015 through March 2021.CHIP was defined as the presence of CH with no cytopenia or morphologic findings of hematologic malignancy, while CCUS was defined as the presence of CH with cytopenia, using the established criteria of persistent anemia (hemoglobin <11 g/dL); neutropenia (absolute neutrophil count <1.5 K/μL); or thrombocytopenia (platelets <100 K/ μL) for at least 4 months. 11,12Excluded were patients, who had morphologic evidence of a myeloid neoplasm, previous history of any myeloid neoplasm, and/or donorengrafted CHIP.Patients' comorbidities were assessed according to the validated Adult Comorbidity Evaluation 27 (ACE-27) score. 13,14

| Assessment of somatic mutations
Genomic DNA from patients' peripheral blood and/or whole bone marrow aspirate samples was sequenced with an NGS platform in a Clinical Laboratory Improvement Amendments (CLIA)-certified laboratory within the Department of Hematopathology at The University of Texas MD Anderson Cancer Center. 15,16

| Outcome definitions and statistical methods
The date of CH diagnosis was defined as the date of the first identification of any somatic mutations.The date of transformation to myelodysplastic syndrome (MDS), chronic myelomonocytic leukemia (CMML), or acute myeloid leukemia (AML) was derived from the date of the first bone marrow evaluation showing morphological evidence of a myeloid malignancy.Overall survival (OS) was calculated from the time of CH diagnosis to the time of death.OS after transformation was calculated from the time of MDS/AML diagnosis to the time of death.Time to transformation was calculated from the time of diagnosis to the time of transformation or death without transformation.The cumulative incidence of transformation was estimated with death without transformation as a competing event.
Among patient characteristics, comparisons between continuous variables were made using the Kruskal-Wallis test, and comparisons between categorical variables were made using the χ 2 test.Paired two-sample t-test and Wilcoxon rank-sum test were used to compare continuous variables before and after transformation to MDS/AML.The Kaplan-Meier product limit method and cumulative incidence function were used to estimate survival outcomes. 17The Cox proportional hazard method was used to analyze association between CH and time to hematologic mutational burdens and anemia.Nevertheless, such patients were less likely to die of their myeloid neoplasm than of primary malignancy or comorbidities.

K E Y W O R D S
clonal hematopoiesis, clonal hematopoiesis of indeterminate potential, clonal cytopenia of undetermined significance, CHIP, CCUS malignancy transformation. 18The associations between CH and cumulative incidence of transformation to a myeloid neoplasm and death without such transformation were analyzed by competing risk regression (i.e., subdistributional hazard model). 19Hazard ratios (HR) and subdistribution hazard ratios (SHR) with 95% confidence intervals (CI) were reported for time-to-event analysis.All analyses were performed using GraphPad Prism 9.0.0 and RStudio Version 4.1.0. 20
Genomic DNA was extracted from bone marrow samples in 70 patients (90%) and peripheral blood in eight individuals (9%).In those who were referred for a myeloid somatic mutation seen on peripheral blood, all underwent complete hematologic work-up, including bone marrow evaluation, except four patients with no evidence of abnormalities on complete blood counts and differentials.The median number of mutations was one (range: 1-8), and the median variant allele frequency (VAF) was 0.078 (range: 0.011-0.729)at diagnosis.The distribution of somatic myeloid mutations is shown in Figure 1.DNMT3A (40%), TET2 (31%), TP53 (26%), and ASXL1 (18%) were the most frequent mutations.Among the 71 patients with available cytogenetic data, 55 (77%) had normal karyotype and seven (10%) had a single chromosomal abnormality.

| Survival and transformation outcomes
With a median follow-up time from diagnosis of 27 months (range: 0.1-79), the median OS (mOS) for the entire group was not reached, with a 2-year survival rate of 79% (95% CI: 70, 90).The CCUS-T cohort had the worst survival outcomes mOS: 32 months (95% CI: 32, not estimable [NE]), whereas the mOS of all other patients was not reached (p = 0.45, Figure S1).There were no differences in survival based by the presence of cytogenetic abnormalities (p = 0.970).Twenty patients (26%) died, of whom seven (35%) died of their primary cancer, four (20%) died of comorbidities, and four (20%) died of myeloid neoplasms following transformation (Table 2).Of the 78 patients, 12 (15%) experienced transformation to MDS/CMML (n = 11) or AML (n = 1).Patient characteristics and outcomes summarized in Table S1.Transformation occurred in seven CCUS-O (21%), one CHIP-T (17%), two CCUS-T (15%), and two CHIP-O patients (8%).The median time to transformation was not reached, and there was no significant difference in time to transformation among groups (p = 0.53, Figure S2).The mOS after transformation was also not reached.Of interest, the median number of mutations (two vs. three, p = 0.67) and VAF (0.36 vs. 0.37, p = 0.82) were similar at the time of CH diagnosis and at transformation, respectively.The most common mutations at transformation were TET2 (64%), ASXL1 (45%), IDH2 (27%), EZH2 (27%), and TP53 (18%).All three patients (two CCUS-O and one CCUS-T) with EZH2 mutations at baseline progressed to MDS. Figure 2 illustrates the genomic evolution from diagnosis to myeloid neoplasm transformation.Two patients (17%) acquired a new cytogenetic abnormality and five patients (42%) acquired new mutations at transformation, with ASXL1 being the most frequently acquired mutation (40%).Six patients (50%) did not receive treatment for their lower-risk MDS or CMML.
The cumulative incidence of transformation was further calculated with death without transformation as a competing event after combining the CHIP-O and CHIP-T subgroups due to small patient numbers.The 3-year cumulative incidence of myeloid neoplasm transformation was higher among the CCUS-O (24%) and CCUS-T (21%) groups than in the CHIP group (6%, p = 0.48, Figure S3).CCUS-T patients had the highest 3-year cumulative incidence of death without transformation risk (53%) compared to the CCUS-O (35%) and CHIP (20%, p = 0.14) groups, which reflected a higher proportion of deaths due to primary malignancy (40%) and comorbidities (40%) in the CCUS-T group.

| Exploratory analysis
Patients were further recategorized into high-risk (H) or low-risk (L) cohorts based on VAF ≥0.2 or non-DTA mutations, which have been reported to portend a higher risk of progression. 21As illustrated in Figure 3A, the 3-year cumulative incidence of myeloid neoplasm transformation was highest in the H-CCUS group (37%) compared to the L-CCUS (8%), H-CHIP (6%), and L-CHIP groups (0%, p = 0.037).Patients were also risk-stratified according to the recently-presented and published clonal hematopoiesis risk score (CHRS), a prediction tool for the risk of progression to myeloid neoplasms in healthy adults with CH. 22 Per the CHRS, 16 patients (21%) were deemed at low risk, 38 (49%) at intermediate risk, and 21 (27%) at high risk of transformation to a myeloid neoplasm.There was no difference in CHRS risk group by previous exposure to chemotherapy, radiation therapy, or autologous stem cell transplantation.When examining particular chemotherapy regimens, alkylating agents were not enriched in a particular risk group (p = 0.380), but interestingly, no CHRS low-risk individual received topoisomerase inhibitors prior to the detection of CHIP or CCUS (p = 0.077).CCUS-T (38%) and CCUS-O patients (33%) exhibited a trend toward a higher risk of myeloid neoplasm transformation than CHIP-T (17%) and CHIP-O (15%) patients, though the difference was not significant (p = 0.24).The 3-year cumulative incidence of transformation was 0% in the low-risk group compared to 15% in the intermediate-risk (p = 0.098) and 28% in the high-risk groups (p = 0.05, Figure 3B).There were no differences in time to transformation or mOS by CHRS risk stratification (Figure S4).When comparing the two risk stratification methods, of the 16 CHRS low-risk patients, 15 were also categorized as L-CHIP/L-CCUS.One CHRS low-risk patient was classified in the high-risk cohort due to the presence of a mutation in BCORL1 that is considered non-DTA but not a high-risk mutation by CHRS.
The patients at a tertiary cancer center differ from the healthy adult population, as they appear to have more high-risk features.Two large population studies in the United Kingdom and the Netherlands showed low transformation rates, with development of myeloid neoplasms 2.37% over a median follow-up time of almost 12 years and a cumulative incidence of a hematological cancer of 2.5% every 5 years. 22,23However, the mutations most frequently seen in these studies were overwhelmingly DNMT3A, TET2, and ASXL1, with TP53 mutations seen in approximately 2%-5% of the patients.Our study revealed TP53 mutations in significantly more patients at 25%, indicating a higher-risk cohort with consequently higher rates of transformation at 15%.Similar to these previous reports, DNMT3A mutations appear to portend an indolent CH course, as no patients with DNMT3A mutations transformed.
The impact of CH on therapy-related myeloid neoplasms is an area of great interest.A recent study comparing 33 patients with CCUS following cytotoxic therapies to therapy-related myeloid neoplasms found a transformation rate of 30%. 24Our study evaluating a different patient population resulted in lower transformation rates, though eight patients (67%) had a history of cancer and six (50%) received antineoplastic therapy.Shah et al. noted 44% of evaluable patients had clonal evolution at the time of transformation, similar to our observations. 24Another group of researchers conducted a multicenter CCUS data registry study evaluating those with a history of cancer, previous cytotoxic therapies, and all others. 25Though rates of transformation to myeloid neoplasms were not reported, patients who had received cytotoxic therapies had inferior mOS to those who did not.Our CCUS-T group only incorporated those on active antineoplastic treatments but showed similar survival patterns.Interestingly, their study had an extremely low incidence of mutations in TP53, which was third-most mutated in our patient cohort.Further identification of the cause of cytopenia in patients with CCUS, such as with CCUS-T vs. CCUS-O, are warranted for risk stratification.
Several studies have shown correlations of CH to comorbidities, including cardiac disease and autoimmune disorders. 4,5,26CH is associated with a high risk of atherosclerotic cardiovascular disease. 2,4,5Anti-inflammatory agents, such as canakinumab in TET2-mutated CH patients in the Canakinumab Anti-Inflammatory Thrombosis Outcomes Study (CANTOS), may reduce CHrelated cardiovascular risk. 27,28Other studies indicate that some types of CH may contribute to inflammatory conditions via T-cell dysregulation and VEXAS syndrome, a potentially fatal autoimmune disorder with relapsing polychondritis. 26,29,30As 20% of deaths in our cohort were attributed to comorbidities, close monitoring of CH patients for extra-hematologic manifestations is paramount.
The identification of CH patients who are at high risk of experiencing transformation to a myeloid neoplasm is another area of critical focus.In addition to the publication discussing VAF ≥0.2 and the presence of non-DTA mutations, 21 the CHRS was recently developed to identify individuals at risk of myeloid malignancies from CH. 22 Prognostic variables include number of mutations, high risk mutations, VAF ≥0.2, age, cytopenia, and increased  10,[31][32][33][34] In our cohort, no low-risk individual received topoisomerase inhibitors though there were no statistically significant differences in CHRS risk group by therapeutic subtypes.In those who eventually developed therapy-related MDS/AML, CH has been discovered at the time of their primary cancer diagnosis, before they began antineoplastic therapy. 6,7It is important that such patients be identified and engaged in discussions about the potential benefits and risks of anticancer treatments as well as possible modifications to therapy.There are several limitations to our study.First, the follow-up time is short at over 2 years.A prospective study with longer follow-up times is needed to further explore these observations.Second, the number of patients included is small at 78 with much variability in baseline characteristics, and only 12 patients transformed to MDS or AML.Therefore, the statistical analysis has limited power in this situation.Moreover, the recently-published World Health Organization (WHO) and International Consensus Classifications (ICC) of myeloid neoplasms require VAF ≥0.02 and higher cutoffs for cytopenia to distinguish between CHIP and CCUS. 35,36We included the few patients with VAF <0.02 as many were diagnosed during the initial development of NGS; with the improvement of high-throughput sequencing techniques, these individuals would likely have WHO/ICC-defined CH with current technologies.We also used diagnostic criteria in MDS for cytopenia as this study was conducted prior to the development of WHO and ICC guidelines for CHIP and CCUS, and our patient population was sicker than that of healthy individuals on which most CH studies are based. 11,12There were five patients who were recategorized from CHIP-O to CCUS-O by the new criteria with no difference in results.Additionally, we grouped patients into four cohorts based on cytopenia and the presence of an additional variable that could account for cytopenia, such as active non-myeloid hematologic malignancies (multiple myeloma and chronic lymphocytic leukemia) or concurrent antineoplastic therapy.Due to paucity of laboratory values in treatment-free intervals, we are unable to determine the fluctuation of blood counts and definitively associate cytotoxic therapy with cytopenia.Lastly, CH with PPM1D mutations has been implicated in therapyrelated myeloid neoplasms, but our myeloid NGS panel did not capture this mutation.
In conclusion, the mOS is not reached in cancer patients with CH after over 2 years of follow-up.Progression to MDS/AML occurred in 15% of patients, but the primary causes of death were more frequently the primary malignancy and complication from another comorbid condition rather than transformation to MDS/AML.Patients with higher mutational burdens, represented by VAF ≥0.2, and anemia were at higher risk of transformation to a myeloid neoplasm.Both close monitoring of extra-hematologic manifestations crucial and further investigation into identifying individuals at high risk of myeloid transformation and developing early therapeutic interventions for these patients are warranted.

F I G U R E 2
Oncoplot of Patients who transformed to MDS/CMML or AML.MDS, myelodysplastic syndrome; CMML, chronic myelomonocytic leukemia; AML, acute myeloid leukemia; UID, unique identifier.
Multivariate analysis of predictors of transformation.The full model includes variables with p < 0.15 on univariate analysis by Cox Regression.bThe reduced model includes significant variables using backward elimination methods.red blood cell indices by red cell distribution width and mean corpuscular volume.In our study investigating predominantly cancer patients, the CHRS was able to discern patients at low-versus intermediate-risk and high-risk of transformation (p = 0.098 and 0.05, respectively) but was less able to distinguish between intermediate-and high- T A B L E 3 a